Methods and compositions for expressing negative-sense viral RNA in canine cells

Abstract

The present invention provides novel canine pol I regulatory nucleic acid sequences useful for the expression of nucleic acid sequences in canine cells such as MDCK cells. The invention further provides expression vectors and cells comprising such nucleic acids as well as methods of using such nucleic acids to make influenza viruses, including infectious influenza viruses.

1 . An isolated nucleic acid comprising a canine RNA polymerase I regulatory sequence.  
     
     
         2 . The nucleic acid of  claim 1 , wherein the regulatory sequence is a promoter.  
     
     
         3 . The nucleic acid of  claim 1 , wherein the RNA polymerase I regulatory sequence comprises nucleotides 1 to 1803 of SEQ ID NO: 1 or a functionally active fragment thereof.  
     
     
         4 . The nucleic acid of  claim 1 ,  2  or  3 , wherein the regulatory sequence is operably linked to cDNA encoding a negative-strand viral genomic RNA or the corresponding cRNA.  
     
     
         5 . The nucleic acid of  claim 4 , wherein the nucleic acid further comprises a transcription termination sequence.  
     
     
         6 . The nucleic acid of  claim 5 , wherein the negative-strand viral genomic RNA is an influenza genomic RNA.  
     
     
         7 . An expression vector comprising the nucleic acid of  claim 6 .  
     
     
         8 . A method for producing an influenza genomic RNA, comprising transcribing the nucleic acid of  claim 6 , thereby producing an influenza genomic RNA.  
     
     
         9 . A method for producing a recombinant influenza virus, comprising culturing a canine cell comprising the expression vector of  claim 7  and one or more expression vectors that express an mRNA encoding one or more influenza polypeptide selected from the group consisting of: PB2, PB1, PA, HA, NP, NA, Ml, M2, NS1, and NS2; and isolating the recombinant influenza virus.  
     
     
         10 . The method of  claim 9 , wherein influenza virus produced is infectious.  
     
     
         11 . The method of  claim 9 , wherein the method results in the production of at least 1×10 3  PFU/ml influenza viruses.  
     
     
         12 . A cell comprising the expression vector of  claim 7 .  
     
     
         13 . The cell of  claim 12 , wherein the cell is a canine cell.  
     
     
         14 . The canine cell of  claim 13 , wherein the canine cell is a kidney cell.  
     
     
         15 . The canine kidney cell of  claim 14 , wherein the canine kidney cell is an MDCK cell.  
     
     
         16 . A method for generating in cultured canine cells a recombinant segmented negative-strand RNA virus having greater than 3 genomic vRNA segments, said method comprising: (a) introducing into a population of canine cells a first set of expression vectors capable of expressing in said cells genomic vRNA segments to provide the complete genomic vRNA segments of said virus; (b) introducing into said cells a second set of expression vectors capable of expressing mRNA encoding one or more polypeptides of said virus; and (c) culturing said cells whereby viral particles are produced.  
     
     
         17 . The method of  claim 16 , wherein infectious influenza viral particles are produced.  
     
     
         18 . A method for generating in cultured canine cells infectious influenza viral particles, said method comprising: (a) introducing into a population of canine cells a set of expression vectors capable of expressing in said cells i) genomic vRNA segments to provide the complete genomic vRNA segments of said virus and (ii) mRNA encoding one or more polypeptides of said virus; (b) culturing said cells whereby said viral particles are produced.  
     
     
         19 . A virus produced by the method of  claim 16 .  
     
     
         20 . The method of  claim 16 ,  17  or  18 , wherein helper virus is used.